Homocysteinemia and Related Pathology
Cardiovascular disease due to atherosclerosis, and arterial and venous thromboembolism are a major cause of mortality and morbidity in North America. Although conventional risk factors such as hypercholesterolemia, hypertension and smoking are associated with increased risk for cardiovascular disease, they do not account for all cases. There is mounting evidence that elevated blood levels of homocysteine (hyperhomocysteinemia) represent a significant risk factor for premature vascular and thrombotic disease. Hyperhomocysteinemia has been found in approximately 20-30% of individuals identified with coronary artery disease, peripheral vascular disease and stroke (Boers et al. (1985) N. Engl. J. Med. 313: 709; Clarke et al. (1991) N. Engl. J. Med. 324: 1149; Sethub et al. (1995) N. Engl. J. Med. 332: 286; den Heijer et al. (1995) Lancet 345: 882. Despite the fact that a number of inherited or acquired conditions can lead to hyperhomocysteinemia, the relationship between increased homocysteine levels and vascular disease is present regardless of the underlying metabolic causes.
The majority of inherited cases are believed to result from deficiency of the enzyme cystathionine .beta.-synthase, which mediates the condensation of homocysteine with serine to form cystathionine. Individuals homozygous for cystathionine .beta.-synthase deficiency suffer from ocular, skeletal and neurologic complications, and are at high risk for premature vascular disease and venous thrombosis (Mudd et al. (1981) Am. J. Hum. Genet. 33: 883; Ueland et al. (1989) J. Lab. Clin. Med. 114: 473; Ueland et al. Plasma homocysteine and cardiovascuar disease. in Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function Francis R B, Ed. (1993) Marcel Dekker, Inc., pp. 183-236). The homozygous form of cystathionine .beta.-synthase deficiency is associated with a 9% incidence of myocardial infarction, a 38% incidence of thromboembolic disease and a 23% incidence of cerebrovascular and frequent peripheral vascular events (Grieco et al. (1977) Am. J. Med. Sci. 273: 120). Despite the autosomal recessive inheritance, there now is evidence for an association between heterozygosity for cystathionine .beta.-synthase deficiency and premature vascular disease (Clarke et al. (1991) op.cit; Boers et al. (1985) op.cit; Malinow et al. (1989) Circulation 79: 1180; Kang et al. (1986) J. Clin. Invest. 77: 1482). Based on the frequency of the homozygous condition at birth, it is estimated that at least 1-2 percent of the population (not including individuals with other different types of acquired or inherited abnormalities of homocysteine catabolism) has hyperhomocysteinemia and are at risk for premature vascular disease and thrombosis.
Although the identification of homozygous cystathionine .beta.-synthase deficiency can be made on the basis of markedly elevated plasma levels of homocysteine, this means of diagnosis is inconvenient and is poorly suited for identifying the more common heterozygous state because plasma homocysteine levels in heterozygous individuals can overlap with those found in healthy individuals (McGill et al. (1990) Am. J. Med. Genet. 36: 45). This problem can be partially circumvented using a methionine-loading test in which L-methionine is given orally after an overnight fast. Venous blood samples are obtained immediately before and four to eight hours after methionine administration for measurement of serum non-protein bound homocysteine. Homocysteine levels are then determined using ion-exchange chromatography. This approach is both time-consuming and uncomfortable for the patient and fails to identify all heterozygotes (Boers et al. (1985) op.cit; Kang et al. (1986) op.cit). Other methods are available to identify heterozygotes but these are even more problematic. For example, the activity of cystathionine .beta.-synthase can be determined from cultured fibroblasts but this requires a skin biopsy, an invasive procedure. Recently, a bacterial screening-expression system has been developed to identify patients with hyperhomocysteinemia caused exclusively by cystathionine .beta.-synthase deficiency (Kozich and Kraus (1992) Hum. Mut. 1: 113). This system also allows for the characterization of the mutation within the cystathionine .beta.-synthase gene. There are, however, at least two major disadvantages with this approach. Firstly, it can only be used to identify individuals deficient in the enzyme cystathionine .beta.-synthase. Therefore, patients with other forms of genetic or acquired conditions which predispose them to hyperhomocysteinemia cannot be identified. Secondly, allelic and genetic heterogeneity within the cystathionine .beta.-synthase gene complicates the reliable use of this approach.
At present, there is no simple and reliable test to identify individuals with hyperhomocysteinemia. This is a serious drawback because some forms of hyperhomocysteinemia can often be corrected by vitamin supplementation (Ubbink et al. (1993) Clin. Invest. 71: 993; Franken et al. (1994) Arteroscler. Thromb. 14: 465. Since such treatment results in only minor side effects, it is important to identify the individuals at risk for hyperhomocysteinemia so that they can be targeted for treatment aimed at reducing plasma homocysteine, thereby reducing the risk of vascular disease. Furthermore a simple diagnostic test would provide a method to monitor the success of such treatment and its impact on subsequent atherosclerosis.
Despite intensive study the exact mechanisms responsible for homocysteine-induced vascular disease remain unclear. However, several recent reports have shown that homocysteine contributes to endothelial cell damage and dysfunction in vitro. These include induction of a protease activator of Factor V (Rodgers and Kane (1986) J. Clin. Invest. 77: 1909), induction of an inhibitor of protein C activation (Rodgers and Conn (1990) Blood 75: 895), aberrant processing and secretion of thrombomodulin (Lentz and Sadler (1991) J. Clin. Invest. 88: 1906) and von Willebrand Factor (Lentz and Sadler (1993) Blood 81: 683), and inhibition of thrombomodulin cofactor activity (Hayashi et al. (1992) Blood 79; 2930). Alteratively, homocysteine may promote the proliferation of vascular smooth muscle cells, a major component in atherosclerotic plaque, while decreasing endothelial cell proliferation (Tsai et al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91; 6369). Marked platelet accumulation at sites of vascular injury and platelet rich occlusive thrombi are also distinctive pathologic features of both human and experimental hyperhomocysteinemia (James et al. (1990) J. Am. Coll. Cardiol. 15: 763; Harker et al. (1983) Circ. Res. 53: 731). In addition, platelet survival appears to be shortened in patients with hyperhomocysteinemia. However, the mechanisms involved in homocysteine-induced platelet dysfunction also remain elusive.
The diagnosis of cardiovascular disease requires a detailed clinical evaluation, and diagnosis generally cannot be made until significant symptoms of coronary artery occlusion and plaque formation are clinically apparent. Thus, there is a need for a simple and reliable diagnostic method for hyperhomocysteinemia, which is associated with a substantial fraction of atherosclerotic disease patients, and for methods to use such tests to identify patients with hyperhomocysteinemia and monitor the effect of pharmacologic interventions on homocysteine-induced pathology.